Small regulatory RNAs participate in many eukaryotic cell functions, microRNAs (miRNAs), the major subclass of small regulatory RNAs in animal species, regulate gene expression post-transcriptionally by destabilizing and/or reducing the translation of specific 'target'mRNAs. Post-transcriptional gene regulation has revolutionary implications, yet very little is known of the roles played by miRNAs in neurons or neurodegenerative disease. AIM 1: CHARACTERIZE THE POLYRIBOSOMAL miRNP IN NEURON-LIKE CELLS. miRNA-related biochemistry is poorly understood. We hypothesize that miRNA-containing polyribosomal ribonucleoprotein complexes (miRNPs) represent the biochemical substrate for miRNA:mRNA regulation. We will partially purify the polyribosomal miRNP from Weri retinoblastoma cells, to characterize the biochemical properties and the protein components of this important particle. AiM 2: IDENTIFY AND CHARACTERIZE miRNA:mRNA PAIRS. Although hundreds of human miRNAs are known, most mRNA targets are unknown. We discerned "rules" that govern miRNA:target mRNA interaction. Using these guidelines our bioinformatician collaborators predict mRNA targets regulated by human miRNAs. We will use cell biological tools to verify hypothesized miRNA-mRNA partners relevant to human neurological diseases. AIM 3: CHARACTERIZE A miRNA INTERACTION THAT MAY REGULATE THE EXPRESSION OF ALPHA-SYNUCLEIN (A-SN). On the basis of preliminary evidence, we hypothesize that an evolutionarily conserved sequence element in the 3'-untranslated region of A-SN mRNA is recognized by a specific miRNA (miR-93). A-SN protein plays a central role in some neurodegenerative diseases and its regulation by a miRNA would have important implications in neurobiology, neurodegenerative disease, and RNA biology. We will study this interaction using neuronal cell lines, as a prototype of miRNA:mRNA validation. We will also extend the analyses to human brain tissue in health and disease.